Sodium and potassium ion-dependent adenosine triphosphatase ((Na+ + K+)-ATPase) is the enzyme in the plasma membranes of all animal cells that is responsible for the primary active transport of sodium out of and potassium into the cytoplasm. It is the effective target of the cardiotonic steroids such as digitalis and digoxin. The sequence of the catalytic alpha-polypeptide of (Na+ + K+)-ATPase has recently become available. Ten hydrophobic segments within this sequence have been designated as candidates for spanning the bilayer of the plasma membrane when the enzyme is in its native structure. It is our objective to determine which of these ten sequences do fulfill this role by determining on which side of the plasma membrane each of the hydrophilic regions between these hydrophobic segments lies. A specific lysine, glutamic acid, or tyrosine in each of these hydrophilic regions has been chosen as a suitable target to be modified by an impermeant reagent. Vesicles of plasma membrane containing high concentrations of (Na+ + K+)-ATPase and sealed in a right-side-out orientation will be the specimens used for each of these modifications. The product of a given modification at one of the targeted amino acids will be isolated by digesting the protein and isolating by immunoadsorption the peptide in which the modified amino acid is located. The immunoadsorbent used for the isolation will be made from antibodies directed against a synthetic peptide containing the amino terminal or carboxyl terminal sequence of the modified peptide. By determining whether each of the targeted amino acids is located on the cytoplasmic or the extra-cytoplasmic surface of the membrane, the topology of the alpha-polypeptide in native (Na+ + K+)-ATPase will be established. This will identify those sequences that span the bilayer and form the central compartment through which potassium and sodium pass in and out of the cell. This information will increase our insight into the mechanism of (Na+ + K+)-ATPase whose function is of central importance to those physiological processes that pace heartbeat, power fluid flows in the kidney and intestine, and create the action potentials of the nervous system.